370

EXTENDED METHYLENE BLUE TEST FOR ANTIBIOTICS IN MILK1

BURDET HEINEMANN

Producers Creamery Company,

Springfield, Missouri
( Received for publication August 3, 1960)
Downloaded from jfoodprotection.org by UNIVERSITY OF PITTSBURGH on 09/24/19. For personal use only.

In order to maintain adequate control over the
presence of antibiotics in milk by the dairy plants,
simplified platform tests are highly desirable. Such
tests should be capable of performance by any lab-
oratory technician and yet be sensitive to low levels of
penicillin or other antibiotics. Several procedures
were studied and the most satisfactory procedure is
presented below. This procedure may be performed
using the same equipment which is required for
Journal of Milk and Food Technology 1960.23:370-372.
cultures and both color change and curd formation as
endpoints.
The results obtained using these methods are given
in Table 1. It will be noted that one-tenth of a unit
of penicillin per ml of milk is not detected when raw
manufacturing grade milk is tested. When raw Grade
A milk is tested to which one-tenth unit penicillin
has been added per ml of milk, 100 per cent of the
samples are detected. When manufacturing milk

making the methylene blue test for grades of milk.
The only additional requirement is the use of a cui-·
ture of S. thet·mophilus, which may be obtained from
commercial laboratories supplying the dairy industry
with freeze-dried cultures.
METHODS AND 0BSERVATIONS
The following methods were studied: (a) the ex•
tended methylene blue test and curd observations
made on raw milk samples using lactic cultures ( 16
hours at 70-72"F.); (b) extended methylene blue
and curd tests on pasteuriZed milk samples using lac-
tic cultures; (c) extended methylene blue and curd
tests on pasteurized milk samples using S. thermo-
phi/us cultures ( 4-6 hours at 98°F.) (d) five-hour
methylene blue tests on pasteurized milk samples
using lactic cultures and color change as an end-
point; and (e) four to eight hour methylene blue tests
on pasteurized milk samples using S. thermophilus
lPrcscnted at the Annual Meeting of the American Dairy
Science Association, Logan, Utah, June 20, 1960.
is pasteurized before the lactic starter is added and
when curd is used as the endpoint, 97 samples out of
102 were detected. Using color change as an end-
point and S. thermophilus as the culture 162 samples
were detected out of 165, and when lack of curd
formation was used as an endpoint all of the samples
were detected. Because of these observations the
test selected involved pasteurizing the samples and
using S. thermophilus as the test organism. It is
based on the method described by Neal and Cal-
bert (1).
The antibiotic test is performed after the comple-
tion of the methylene blue test described in Standard
Methods ( 2). When grading manufacturing milk
by the methylene blue test the No. 4 grade tubes are
removed, identified, and placed in an ice bath. When
the No. 3 tubes have reduced, these also are placed
in an ice bath after identifying each tube. At the
completion of the 3~ hour reading, the No. 4 and
No. 3 tubes are returned to their original position in
the tray. At this time control samples with and
without added antibiotics containing 10 ml of milk
and 1 ml of methylene blue solution are placed in

TABLE 1- EFFECT OF METHOD ON DETECTION OF 0.1 UNIT PENICILLIN G. ADDED PER ML MILK

Ra.w Number detected

Method• Type milk Starter or No. samples Incubation by observation of
past. conditions Color (blue) Curd (none)

1 Grade A s. lactis Raw 94 16 hrs/72°F. 94 84

1 Mfg. s. lactis Raw 102 16 hrs/72°F. 0 10

2 Mfg. s. lactis Past. 102 16 hrs/72°F. 3 97
3 Mfg. S. therm. Past. 165 4-6 hrs/98°F. 162 165
4 Mfg. S. lactis Past. 56 5 hrs/98°F. 47

5 Mfg. S. therm. Past. . 56 4-8 hrs/98°F. 56 56

 METHYLENE BLuE TEST FoR ANTIBIOTICS 371

TABLE 2 - EFFECT OF PASTEURIZING TIME AT 180"F. ON milk may be placed in the methylene blue water bath
REDUCTION TrME IN HouRS UsiNG S. thermophilus; AvERAGE and brought to temperature. rThe drys. thermophilus
OF 4 TRIALS (AFTER COMPLETION OF METHYLENE
culture may then be added to the milk on the night
BLUE TEST FOR GRADE)
prior to testing for antibiotics. The culture is incu-
Minutes held at 180°]<', lteductlon times when units of bated at 98°F. overnight and is then ready for use
penicillin G added per ml were :
in the morning. Experience has shown that this
0.0 0.05 0.10
procedure results in a more nearly uniform culture
0 5.4 7.3 7.3 from month to month. It also eliminates the need
1 6.7 10.8 11.5 to carry S. thermophilus cultures in the plant laf?ora-
Downloaded from jfoodprotection.org by UNIVERSITY OF PITTSBURGH on 09/24/19. For personal use only.

2 6.2 10.3 12.5+ tory.

3 6.4 9.5
6 6.2 12.1
one of the trays and the trays then placed in water
at a temperature of 180°F. for a total period of 2
minutes.
Table 2 shows that pasteurization of the milk
Journal of Milk and Food Technology 1960.23:370-372.
10.1 Table 4 indicates that the small quantities of S.
9.1 thermophilus are more sensitive to penicillin. The
differences, however, are not great and it was found
that 0.5 ml resulted in slightly more uniform rates of
methylene blue reduction.
In order to become familiar with the test, prelimi-
nary trials should be made on milk samples to which
known quantities of antibiotics have been added.

samples is necessary but that excessive holding time For this purpose, infusion kits may be obtained from
tends to reduce the sensitivity of the test. For this drug stores handling veterinarian supplies. Stand-
reason a total time of 2 minutes at 180°F. was selected ards may be prepared containing per ml of milk, 0.1
as the pasteurization conditions. unit of penicillin or 1 microgram of terramycin or
A sample of milk taken from a large volume of aureomycin. Several brands have been tested and
mixed milk in a storage tank may be used as the con- found satisfactory. It also has been found that pre-
trol or any sample of milk known to be free from in- pared milk samples containing added antibiotics may
hibitors. Mter pasteurization the trays must be be held frozen in test tubes for at least 2 months.
placed immediately in ice water and may be held These test tubes, containing 10 ml of milk, may be
overnight at 35°F. to 40°F. for completion in the removed and used as positive controls every time
morning. If nonfat dry milk is used as a control, it a series of test~ is run.
should not be pasteurized after reconstituting.
The same procedure as above may be followed TABLE 3 EFFECT OF STRAIN OF S. thermophaus ON
METHYLE.."'E BLUE REDUCTION TIME
using the Grade A grading system and pasteurizing
the tubes after 5~ hours in the methylene blue water Hours Reduction Time
bath. If methylene blue grading is not required, this Strain No. No penicillin added 0.1 unit penicillin G/ml
portion of the test may be eliminated and the 10-ml
1 3.16 6.24
samples simply pasteurized after collection.
2 4.83 6.23
The next morning the samples are warmed to 98°F., 3 5.00 6.25
0.5 ml of an active culture of S. thermophilus added 4 5.00 5.83
to each tube and the tubes mixed. One ml of meth- 5 4.75 6.33
ylene blue solution is then added to each tube regard- 6 2.92 3.23
less of whether it has reduced or not on the methyl- 2.92 4.17
7
ene blue grade test and the tubes again mixed. The
8 7.00 8.58
trays are then placed in the water bath at 98°F. for
9 4.92 9.88
3}f hours or for a period of Jf hour after ·the control
containing no inhibitor has reduced. Read all tubes
which are still blue at this time as being positive for TABLE 4 EFFECT OF AMoUNT OF S. Thermophilus ADDED
bacterial inhibitors. 0::\1 REDUCTION TThfE IN HoURS; A VERACE OF 12 TRIALS
Table 3 shows that various strains of S. thermo- (AFTER CoMPLETING METHYLENE BLUE TEST FOR GRADE)

philus vary in their sensitivity to penicillin. Strain

'I'lme to reduce (hours)
No. 6 was not satisfactory and strain No. 9 was the
Amount added 0.0 Unit• 0.05 Unit• O.Hl Unit•
culture which was used in these eA.-periments.
Dry cultures may be obtained from commercial 1 drop 3.96 9.25 8.58
laboratories providing penicillin sensitive cultures are 0.5 ml 3.69 7.00 6.67
requested. Canned sterile culture milk is also avail- ~····- ,------'---------------
able commercially. For convenience the sterile starter •Units of Penicillin G added per ml
 372 METHYLENE BLUE TEST FoR ANTimoncs
The extended methylene blue test as described will
detect 0.05 unit Penicillin G per ml of milk and will
detect many samples containing as little as 0.02 unit
per mi. Aureomycin and terramycin may be detected
at levels of 0.5 to 1 microgram per mi. Streptomycin
may be detected at levels of 3 to 5 micrograms per
mi. As a confirmatory test, after the methylene blue
has reduced, all tubes may be held for a period up to
10 hours and observed for curd formation. Samples
Downloaded from jfoodprotection.org by UNIVERSITY OF PITTSBURGH on 09/24/19. For personal use only.
containing no antibiotic set up a firm curd well ahead
of those samples which contain at least 0.05 unit of
Penicillin G per ml. of milk.
11
THE PROTECTIVE SCREE~" PROGRAM FOR CA~~ED FOODS1
M.P. VERHULST
Journal of Milk and Food Technology 1960.23:370-372.
Wisconsin Canners Association,
Tenney Building, Madison, Wi8conson
The canning industry has long been aware of the
fact that in the maintenance of consumer confidence
in canned foods, each member of the industry is "his
brother's keeper." One of the principal reasons for
organizing the National Canners Association some
fifty years ago was to insure the safety and whole-
someness of canned foods. Perhaps some can re-
member the botulism scare in connection with can-
ned ripe olives in the early 1920's. This hurt canners
of every product, and although no deaths from botu-
linus poisoning in commercially canned foods have
occurred within the past twenty-five years, it took
a long time to rebuild consumer confidence in can-
ned food.
The cranberry episode last November was another
dramatic demonstration of how all the members of
the cranberry industry could be injured by the acts
of a few; and some Wisconsin cranberry canners
were among those injured.
Before the 1960 canning season got under way,
the canning industry laid plans to prevent the occur-
rence of a similar situation occurring in connection
with any other canned food. For want of a better
name, we have called this program "The Protective
Screen Program for Canned Foods." It amounts to
a series of defenses to protect canned foods against
chemical contamination.
The basic program was developed by the National
Canners Association and is being carried out in co-
1Presented at the annual meeting of the Wisconsin Associa-
tion of Milk and Food Sanitarians, at Elkhart Lake, Wisconsin
September 13, 1960. · '
ACKNOWlEDGEMENTS
The helpful suggestions of Dr. J. C. Flake and the assist-
ance of Mr. Ben Coble and members of his staff in perform-
ing a large number of tests is greatly appreciated. Thanks
are also due to Mr. Neil Angevine for supplying several dried
cultures of S. thermophilus.
REFERENCES
L Neal, C. E. and Calbert, H. E. The use of 2, 3, 5-
Triphenyltetrazolium chloride as a test for antibiotic sub-
stances in milk J. Dairy Sci., 38: 629-633. 1955.
2. Standard Methods for the Examination of Dairy Products,
lOth ed. Am. Pub. Health Association, Inc. New York, N. Y.
1953.
operation with state canners' associations. Of course,
the success of the program depends primarily on the
care and intelligence with which the individual can-
ner utilizes the program.
The protective screen program is directed to the
problem of food additives as well as to pesticide
residues and is broken down into three phases:
I.To prevent contamination of our raw product.
II. To prevent the processing of any contaminated
raw product.
III. To prevent the addition of illegal chemicals
during processing.
The features of each of these are as follmvs:
I. To prevent the contamination of our raw product.
This is the most important part of the program and
involves six steps:
A. All canners, not only the members of the Na-
tional Canners Association, are furnished by that as-
sociation complete and current information on what
agricultural chemicals have been accepted for regis-
tration by the U. S; Department of Agriculture under
the Federal Insecticide, Fungicide and Rodenticide
Act. Lists of these' pesticides were distributed early
this year and are kept up to date by means of a
special Pesticide News Letter. These lists, of course,
include not only insecticides, fungicides and rodenti-
cides, but also herbicides, fumigants, defoliants, des-
sicants, and plant growth regulators.
B. All canners were urged to furnish each of his
growers a list of pesticides to be used on the specific
crop or crops to be produced by the grower, together
with directions and limitations as to the use of such
pesticides. A few canners prepared such lists of their